Interferons (referred to as IFN hereinafter) so far known can be classified into three groups, i.e., IFN-.alpha., IFN-.beta. and IFN-.gamma.. IFN-.alpha. is mainly produced from leukocytes, IFN-.beta. from fibroblasts and IFN-.gamma. from T-lymphocytes. These IFNs have been noted as biologically active substances having anti-virus activity, activating activities upon natural killer cells and macrophages, antitumor activity, and the like.
As for IFN-.gamma., there has been a report that it has a stronger cell-inhibiting activity than other IFNs based on the experiment using animal cells [B. Y. Rubin and S. L. Gupta: Proc. Natl. Acad. Sci., USA, 77, 5928-5932 (1980)]. Furthermore, cloning of an IFN-.gamma. cDNA and determination of its base sequence were recently reported [P. W. Gray, et al.: Nature 295, 503 (1982), R. Devos, et al.: Nucleic Acids Research 10, 2487 (1982)].
The present inventors have independently cloned a cDNA coding for a novel IFN-.gamma. wherein, as apparent from the base sequence illustrated in Table 1, the ninth amino acid of the mature IFN-.gamma. reported by Devos, et al., Lysine (Lys) (AAA), is replaced with glutamine (Gln) (CAA). Further, the IFN-.gamma. cDNA was incorporated into vector pKYP-10 having a tryptophan promoter (Japanese Published Unexamined Patent Application No. 110600/83) and mass production of the IFN-.gamma. in Escherichia coli has been achieved.
Thereafter, the present inventors have studied the production of derivatives of IFN-.gamma. polypeptide using the IFN-.gamma. cDNA illustrated in Table 1 as a starting material.
It was reported that deletion of 11 amino acids from the C-terminal of IFN-.alpha. decreased specific activity to one-third [A. E. Franke, et al.: DNA 1, 223-230 (1982)], whereas addition of 18 amino acids to the N-terminal of IFN-.alpha. did not change specific activity [R. M. King, et al.: J. Gen. Virol. 64, 1815-1818 (1983)].
The present inventors have found that deletion of 5 amino acids from the N-terminal of IFN-.beta. decreased specific activity to about 1/100 [T. Nishi, et al.: DNA 2, 265-273 (1983] and addition of 7 amino acids to the N-terminal of IFN-.beta. decreased specific activity to about 1/10 [S. Itoh, et al.: DNA 3, 157-165 (1984)].
The present inventors have constructed a derivative of IFN-.gamma. wherein the third amino acid of IFN-.gamma. illustrated in Table 1, cysteine (Cys), was replaced with tyrosine (Tyr) (referred to as 3-Tyr-IFN-.gamma. hereinafter) and found that the specific activity was 2-4 times as strong as that of the parent IFN-.gamma.. Further, the derivatives wherein the Cys at the position 1 was replaced with serine (Ser) (1-Ser-IFN-.gamma.), the Cys at the position 3 was replaced with Ser (3-Ser-IFN-.gamma.), the Cys at the positions 1 and 3 were replaced with Ser (1,3-Ser-IFN-.gamma.) and N-terminal amino acids of IFN-.gamma. illustrated in Table 1 were deleted were constructed. Equivalent or more interferon activity was detected for all the derivatives compared with the starting IFN-.gamma..
It has been known that murine mature IFN-.gamma. consists of 136 amino acids, which number is less by 10 than that of amino acids of human IFN-.gamma., and is more heat-stable than human IFN-.gamma.. Murine IFN-.gamma. has Cys at the position 136 as well as at the positions 1 and 3 and these Cys form disulfide bonds in the molecule, which are considered to contribute to stabilization of IFN [D. Goeddel, et al.: Proc. Natl. Acad. Sci. USA 80, 5842-5846 (1984)].
The present inventors have prepared the molecular model of IFN-.gamma. and assumed that the formation of disulfide bond in the molecule of human IFN-.gamma. was possible by introducing Cys between the positions 135 and 138 from the N-terminal, preferably at the position 137. Several derivatives of IFN-.gamma. wherein C-terminal amino acids were deleted and Met at the position 137 was replaced with Cys have been found to have increased heat stability and specific activity.